Parathyroid hormone (PTH) and its importance in regulating the concentration of calcium ions in the blood are well-known. In this regard, such hormone is created by the parathyroid glands and, in combination with other factors, functions to regulate the blood calcium ion levels such that the same is maintained in a steady concentration in both cells and surrounding fluids. Essentially, PTH functions to release stored calcium in the body when serum calcium levels decrease. On the other hand, such secretion is suppressed to the extent the serum calcium concentration increases.
In its complete form PTH comprises a unique peptide comprised of 84 amino acids. The specific sequence of PTH, as provided for a plurality of species, namely, humans, rats, mice, bovids, dogs and pigs, are depicted in FIG. 1, and a variation thereof in FIG. 2, which illustrates the relatively consistent structure such hormone maintains between such identified species.
Given its significance in calcium metabolism for not only humans, but a variety of species, accurately measuring PTH has and continues to be of substantial clinical significance. As is well-documented, serum PTH levels serve as an important parameter for patients having diseases such as hypercalcemia, primary hyperparathyroidism and osteoporosis, among many others. PTH likewise becomes of substantial clinical importance in patients afflicted with chronic renal failure who, because of an excess in PTH production, can develop renal osteodystrophy.
Notwithstanding its important role in metabolism and clinical significance, substantial difficulties have and continue to exist with regard to determinating circulating biologically active PTH levels. First of all, it is well-known that PTH is normally present at extremely low levels, which are normally between 10 pg/ml to 65 pg/ml. Furthermore, it is known that the PTH peptide can be present in a variety of circulating PTH fragments, and in particular large non-(1-84) circulating PTH fragments which appear to co-migrate chromatographically with the (7-84) PTH molecule and are known to significantly interfere with conventional PTH assay measurements. Indeed, the large non-(1-84) PTH fragments may represent about one-half (2) of the PTH measured by a majority of current assays. Exemplary of the current shortcomings of the accurate measurement of PTH are set forth in published Patent Cooperation Treaty International Application No. PCT/US00/00855, International Publication No. W0/00/42437, entitled Methods for Differentiating and Monitoring Parathyroid and Bone Status Related Diseases, and Lepage, Raymond et al., A Non-(1-84) Circulating Parathyroid Hormone (PTH) Fragment Interferes With Intact PTH Commercial Assay Measurement In Uremic Samples, Clinical Chemistry 44:4, 1998 pages 805-809, the teachings of which are expressly incorporated herein by reference.
In an attempt to address such shortcomings, a new assay for detecting PTH levels was introduced by Scantibodies Laboratory, of Santee, Calif., which incorporates a tracer antibody having a binding specificity for the very end N-terminal fraction of human PTH, and more specifically, the first six amino acid residues thereof. As presently understood, such assay appears to minimize cross-reactivity with large non-(1-84) PTH fragments. However, to derive such antibodies requires substantial effort and expense in purifying the same. Moreover, such tracer antibodies have maximum recognition for only the first amino acid residue of PTH, and substantially reduced specificity for any subsequent residues thus obviating its use for some other species where the first amino acid is different. Such drawbacks are discussed in the article by John, M. R. et al., entitled A Novel Immunoradiometric Assay Detects Full-Length Human PTH but not Amino-Terminally Truncated Fragments: Implications for PTH Measurements in Renal Failure, The Journal of Clinical Endocrinology & Metabolism, Vol. 84, No. 11, 1999, p. 4287-4290, the teachings of which are expressly incorporated herein by reference.
Thus, there has been and continues to be a long felt need in the art for an assay binding partner and method of generating the same that is specific for bioactive intact PTH that can determine PTH levels With mitigated cross-reactivity to PTH peptide fragments. There is likewise a need in the art for improved PTH binding partners that can measure PTH levels in a more cost-effective manner and have a greater affinity for PTH that can be readily incorporated into immunoassay kits and the like. Still further there is a need for binding partners, namely, antibodies having a binding recognition that is specific to PTH and can be utilized to detect PTH levels over a wide-variety of species. Finally, there is a need in the art for an improved binding partner having a high binding affinity for PTH that may be readily derived using conventional mechanisms that requires minimal purification, results in greater binding recognition for intact PTH, possesses minimal cross reactivity to large non-(1-84) PTH fragments, and can be derived utilizing methods that generate higher antibody yields than prior art binding partners.